Anal. Chem. 2001, 73, 1849-1854
Ion-Selective Electrode for Transmembrane pH Difference Measurements Takashi Katsu,*,† Hideki Nakagawa,† Tatsuaki Kanamori,‡ Naoki Kamo,‡ and Tomofusa Tsuchiya†
Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan, and Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060-0812, Japan
A triethylammonium-sensitive electrode was constructed using sodium tetrakis[3,5-bis(2-methoxyhexafluoro-2propyl)phenyl]borate as an ion-exchanger and benzyl 2-nitrophenyl ether as a solvent mediator in a poly(vinyl chloride) membrane matrix and was used to determine the pH difference across a cell membrane. The method is based on monitoring of the pH gradient-induced uptake of triethylammonium in situ. The triethylammonium electrode exhibited a near-Nernstian response to triethylammonium in the concentration range of 5 × 10-6-1 × 10-2 M with a slope of 58.5 mV per concentration decade in a buffer solution composed of 150 mM NaCl and 10 mM NaH2PO4/Na2HPO4 (pH 7.5). The limit of detection was 1 µM. In experiments using liposomes, the uptake of triethylammonium into liposomes was quantitatively induced according to the pH difference across the liposomal membrane. The transmembrane pH differences in Escherichia coli cells and the light-induced pH differences across the envelope vesicles of Halobacterium halobium were successfully determined by the present method. Ion-selective electrodes are now commonly used to analyze cellular membrane functions for cation-sugar cotransport1,2 and membrane potential.3,4 These uses of ion-selective electrodes are attractive, because these methods are capable of monitoring a reaction process in turbid cell suspension on a continuous basis without any sampling or separation. We are interested in applying the ion-selective electrode to determine the pH difference across a cell membrane (∆pH) and recently developed a methylammonium-selective electrode for this purpose.5 This method seems to be superior to a tracer-labeled methylamine method,6-9 because * Corresponding author: (phone) +81-86-251-7955; (fax) +81-86-251-7926; (email)
[email protected]. † Okayama University. ‡ Hokkaido University. (1) Tsuchiya, T.; Wilson, T. H. Membr. Biochem. 1978, 2, 63-79. (2) Tsuchiya, T.; Oho, M.; Shiota-Niiya, S. J. Biol. Chem. 1983, 258, 1276512767. (3) Kamo, N.; Racanelli, T.; Packer, L. Methods Enzymol. 1982, 88, 356-360. (4) Demura, M.; Kamo, N.; Kobatake, Y. Biochim. Biophys. Acta 1987, 894, 355-364. (5) Katsu, T.; Akagi, M.; Hiramatsu, T.; Tsuchiya, T. Analyst 1998, 123, 13691372. (6) Rottenberg, H. Methods Enzymol. 1979, 55, 547-569. (7) Roos, A.; Boron, W. F. Physiol. Rev. 1981, 61, 296-434. (8) Azzone, G. F.; Pietrobon, D.; Zoratti, M. Curr. Top. Bioenerg. 1984, 13, 1-77. 10.1021/ac001090t CCC: $20.00 Published on Web 03/16/2001
© 2001 American Chemical Society
no radiolabeling is needed. However, the electrode suffered significant interference from K+ and required pretreatment of cells to measure ∆pH.5 A prerequisite for applicability of an organic amine for ∆pH measurement is that it must be permeable in its neutral form but impermeable in its charged form.6-9 Thus, all amines, except for the quaternary amines that have no neutral form and the lipophilic amines that can permeate through membrane even in their charged form, can basically be used as probes for ∆pH measurement. We chose triethylamine in the present study, because the triethylammonium electrode, which shows little interference by inorganic cations such as Na+ and K+, was expected to be constructed with a combination of a lipophilic ion-exchanger, sodium tetrakis[3,5-bis(2-methoxyhexafluoro-2-propyl)phenyl]borate (NaHFPB), and an appropriate solvent mediator, mimicking several organic ammonium-sensitive electrodes developed previously.10,11 Furthermore, the charged triethylammonium form was thought to be unable to pass through the cell membrane, because the more lipophilic tetraethylammonium ion with one more methyl group than the triethylammonium ion has been reported to be impermeable through liposomal and biological membranes.12,13 Using the triethylammonium electrode, we succeeded in determining ∆pH formed in artificial liposomes, the transmembrane pH differences in Escherichia coli cells, and the light-induced pH differences across the envelope vesicles of Halobacterium halobium. THEORY The principle of ∆pH measurement is that, at equilibrium, the concentration of the neutral form of triethylamine becomes identical on both sides of the membrane, leading to the following relationship:
[H+]in/[H+]out ) [(C2H5)3NH+]in/[(C2H5)3NH+]out
(1)
where the subscripts “in” and “out” mean inside and outside the membrane, respectively. Thus, the pH difference across the membrane (∆pH), defined as pHin - pHout, can be expressed as (9) Rottenberg, H. Methods Enzymol. 1989, 172, 63-84. (10) Katsu, T.; Mori, Y.; Furuno, K.; Gomita, Y. J. Pharm. Biomed. Anal. 1999, 19, 585-593. (11) Katsu, T.; Akagi, M. Anal. Lett. 1998, 31, 55-66. (12) Katsu, T.; Sanchika, K.; Okazaki, H.; Kondo, T.; Kayamoto, T.; Fujita, Y. Chem. Pharm. Bull. 1991, 39, 1651-1654. (13) Katsu, T. Anal. Chem. 1993, 65, 176-180.
Analytical Chemistry, Vol. 73, No. 8, April 15, 2001 1849
follows:
∆pH ) - log
[(C2H5)3NH+]in
(2)
[(C2H5)3NH+]out
This equation means that, under conditions where pHout is higher than pHin, triethylamine in the external medium is concentrated into the cell until the triethylammonium concentration ratio inside and outside the cell reaches ∆pH. Thus, ∆pH can be estimated by measuring the triethylammonium concentration ratio inside and outside the cell. The dissociation constant, pKa, of triethylammonium (10.7 at 25 °C)14 is much higher than physiological pH (around 7.5), and therefore, most of the amine is in the charged form and the total triethylamine concentration is for all practical purposes equivalent to that of the charged amine concentration, which can be measured using the triethylammonium electrode. The triethylammonium concentration inside cells was estimated from the decrease in the extracellular triethylammonium concentration. Thus, ∆pH was calculated from the following equation proposed previously:5
∆pH ) - log
[V +v v10
(E1 - E2)/S
-
]
V v
(3)
where V and v represent outer medium volume and intracellular volume, respectively; E1 is initial potential at 100 µM triethylammonium setting in this experiment; E2 is electric potential after pH gradient was formed; and S is the slope of the triethylammonium electrode. EXPERIMENTAL SECTION Materials. The reagents were obtained from the following sources: triethylamine hydrochloride was from Tokyo Kasei (Tokyo, Japan); NaHFPB was from Dojindo Laboratories (Kumamoto, Japan); benzyl 2-nitrophenyl ether was from Fluka (Buchs, Switzerland); poly(vinyl chloride) (PVC; degree of polymerization, 1020) was from Nacalai Tesque (Kyoto, Japan); egg phosphatidylcholine (PC), egg phosphatidylethanolamine (PE), and egg phosphatidylglycerol (PG) were from Lipid Products (Red Hill, Surrey, U.K.); cholesterol was from Sigma (St. Louis, MO); 2,2,6,6tetramethylpiperidone-N-oxyl (TEMPONE) was from Molecular Probes (Eugene, OR); and potassium tris(oxalate)chromate trihydrate was from Aldrich (Milwaukee, WI). All other chemicals were of analytical reagent grade. Electrode System. A triethylammonium-sensitive electrode was constructed using a PVC-based membrane.10,11 The components of the membrane were 0.1 mg of NaHFPB, 60 µL (∼70 mg) of benzyl 2-nitrophenyl ether, and 30 mg of PVC. The materials were dissolved in tetrahydrofuran (∼1 mL) and poured into a flat Petri dish (28-mm diameter). Then, the solvent was evaporated off at room temperature. The resulting membrane was excised and attached to a PVC tube (4-mm o.d., 3-mm i.d.) with tetrahydrofuran adhesive. PVC membranes containing other solvent mediators were similarly prepared using 0.1 mg of NaHFPB, 60 µL of solvent mediator, and 30 mg of PVC. Each PVC tube was (14) Dean, J. A., Ed. Lange’s Handbook of Chemistry, 11th ed.; McGraw-Hill: New York, 1973.
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filled with an internal solution composed of 1 mM triethylamine hydrochloride and 10 mM NaCl, and the sensor membrane was conditioned overnight. The electrochemical cell arrangement was Ag,AgCl/internal solution/sensor membrane/sample solution/1 M NH4NO3 (salt bridge)/10 mM KCl/Ag,AgCl. The electromotive force (emf) between the silver/silver chloride electrodes was measured using a voltmeter with high input impedance produced by a field-effect transistor operational amplifier (LF356; National Semiconductor, Sunnyvale, CA; input resistance >1012 Ω) and recorded. The detection limit was defined as the intersection of the extrapolated linear regions of the calibration graph.15 The selectivity coefficients of the electrode, kPot i,j , were determined by the separate solution method15,16 using the respective chloride salts at 10 mM and calculated from the equation 1/zj log kPot i,j ) (Ej - Ei)/S + log ci - log cj
where Ei and Ej represent the emf readings measured for triethylammonium and the interfering ion, respectively; S is the theoretical slope of the electrode for triethylammonium (59.2 mV at 25 °C); ci and cj are the concentrations of triethylammonium and the interfering ion, respectively; and zj is the charge of the interfering ion. The electrode was stored in 1 mM triethylamine hydrochloride and 10 mM NaCl when not in use. All measurements were performed at room temperature (-∼25 °C). Preparation of Liposomes and ∆pH Measurements. Liposomes were prepared using the reversed-phase evaporation method5 as follows. Aliquots of lipid stock solutions containing egg PC (10 µmol, 7.7 mg) and cholesterol (7.5 µmol, 2.9 mg) dissolved in chloroform/methanol (1:2, v/v) were placed in a centrifuge test tube (10 mL; Nichiden-Rika, Kobe, Japan). The solvent was evaporated using a centrifugal evaporator (RD400; Yamato, Tokyo, Japan), and the residual lipid was dried under vacuum for several hours. The lipid was then dissolved in 1.5 mL of diethyl ether, followed by the addition of 1 mL of an aqueous solution composed of 50 mM NaCl and 100 mM NaH2PO4/Na2HPO4 (pH 7.5). The mixture was sonicated (5201; Ohtake Works, Tokyo, Japan) at 50 W for 1 min at 0 °C to obtain a homogeneous emulsion. Immediately, it was transferred to a round-bottom flask (20 mL), and the diethyl ether solvent was removed using a conventional rotary evaporator under reduced pressure (using an aspirator) at 25 °C. After the diethyl ether was completely removed by passing nitrogen gas through the mixture, a homogeneous suspension of liposomes was formed. The liposomes were centrifuged (105000g, 20 min) and washed once with 150 mM NaCl and 10 mM NaH2PO4/Na2HPO4 (pH 7.5) to lower the buffer capacity of the outer medium of the liposomes. The final pellet was suspended in 1 mL of the above washing solution. The osmotic pressures of the inner and outer aqueous solutions were measured with an OS osmometer (Fiske, Needham, MA); both were ∼300 mOsm. Liposomes made from egg PE (24 µmol, 17.5 mg)/egg PG (6 µmol, 4.7 mg), and the lipid extracts from E. coli cells (8 mg) were similarly prepared. E. coli lipid was extracted according to the method described previously.17 (15) Buck, R. P.; Lindner, E. Pure Appl. Chem. 1994, 66, 2527-2536. (16) Bakker, E.; Pretsch, E.; Bu ¨ hlmann, P. Anal. Chem. 2000, 72, 1127-1133. (17) Katsu, T.; Yoshimura, S.; Tsuchiya, T.; Fujita, Y. J. Biochem. 1984, 95, 16451653.
The procedure used to evaluate ∆pH depending on the external pH was as follows. The appropriate volume of the liposome suspension prepared above was pipetted and diluted in an assay solution containing 150 mM NaCl and 10 mM NaH2PO4/Na2HPO4 (pH 7.5) to make a volume of 490 µL. Then, 10 µL of 5 mM triethylamine hydrochloride was added to adjust the initial triethylammonium concentration of the liposome suspension to 100 µM. The final volume was 500 µL. The final lipid concentrations of liposome suspensions containing egg PC/ cholesterol, egg PE/egg PG, and the lipid extracts from E. coli cells were 3.8, 13.3, and 4.9 mg of lipid/mL, respectively. The triethylammonium and reference electrodes were immersed in each liposome suspension, along with a miniaturized pH glass electrode (1826A-06T; Horiba, Kyoto, Japan), to simultaneously monitor the external pH of the solution. The suspension was constantly stirred with a stir bar. The present electrode system, including the reference electrode,18 was compact, and therefore, an assay solution volume as low as 500 µL could be examined. The pH of the outer medium was changed by addition of a small amount of 160 mM sodium hydroxide or 160 mM hydrochloric acid. The internal volumes of liposomes were evaluated by the spin label method using a combination of the membrane-permeable spin label TEMPONE and an impermeable broadening agent (potassium tris(oxalate)chromate) as described previously.5,19 The intravesicular volumes of liposomes made from egg PC/cholesterol, egg PE/egg PG, and the lipid extracts from E. coli cells were 11.8, 3.4, and 9.0 µL/mg of lipid, respectively. Preparation of E. coli Cells and ∆pH Measurements. E. coli W3133-2, a derivative of K-12, was used. Cells were grown in minimal salt medium, supplemented with 1% polypeptone, at 37 °C under aerobic conditions.17 Cells were harvested during the exponential phase of growth, washed twice with buffer (150 mM NaCl and 10 mM NaH2PO4/Na2HPO4, pH 7.6), and suspended in the same buffer at 40 mg of cell protein/mL. The protein content was determined by the method of Lowry et al.20 The cell suspension was diluted in an assay solution containing 150 mM NaCl, 10 mM sodium lactate, and 10 mM NaH2PO4/Na2HPO4 (pH 7.6), and then triethylamine hydrochloride was added to adjust the initial triethylammonium concentration in the cell suspension to 100 µM, to create conditions similar to those in the liposome experiments. The final protein concentration of E. coli cells was 12.8 mg of cell protein/mL. An initial external pH of 7.6 was regarded as the internal pH of E. coli cells.21-23 An internal volume of the E. coli cells of 3.7 µL/mg of cell protein24 was used in this study. Preparation of H. halobium Envelope Vesicles and LightInduced ∆pH Measurements. The strains of H. halobium used (18) Katsu, T.; Kobayashi, H.; Fujita, Y. Biochim. Biophys. Acta 1986, 860, 608619. (19) Vistnes, A. I.; Puskin, J. S. Biochim. Biophys. Acta 1981, 644, 244-250. (20) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265-275. (21) Shimamoto, T.; Inaba, K.; Thelen, P.; Ishikawa, T.; Goldberg, E. B.; Tsuda, M.; Tsuchiya, T. J. Biochem. 1994, 116, 285-290. (22) Padan, E.; Zilberstein, D.; Rottenberg, H. Eur. J. Biochem. 1976, 63, 533541. (23) Slonczewski, J. L.; Rosen, B. R.; Alger, J. R.; Macnab, R. M. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 6271-6275. (24) Shiota, S.; Yamane, Y.; Futai, M.; Tsuchiya, T. J. Bacteriol. 1985, 162, 106109.
were S9 and KY-4. S9 contains bacteriorhodopsin and halorhodopsin,25 while KY-4 isolated from strain S9 has halorhodopsin alone.26 The cells were grown in peptone medium, and envelope vesicles were prepared by sonication as described by Lanyi and MacDonald.27 The sidedness of the vesicles was checked by NADHmenadione reductase activity, and all preparations showed 8590% right-side-out vesicles.27 The vesicles prepared from S9 and KY-4 were suspended in test tubes (5 mL; Nichiden-Rika, Kobe, Japan) containing an assay solution composed of 4 M NaCl and 10 mM NaH2PO4/Na2HPO4 (pH 7.5), and then triethylamine hydrochloride was added to adjust the initial triethylammonium concentration in the vesicle suspension to 100 µM. The final volume was 500 µL. The final protein concentrations of S9 and KY-4 vesicles were 14.6 and 13.6 mg of protein/mL, respectively. The test tube was put in a special glass vessel constructed for the thermostating of test solution. Illumination was provided through a cutoff filter (>520 nm) (Y52; Toshiba, Tokyo, Japan) with a 1-kW tungsten projector lamp (Master Hilux H-130; Rikagaku Seiki, Tokyo, Japan). Light intensity at the front of the test tube was 750 W/m2, which was measured with a Kettering radiant power meter (model 4090; Yellow Springs, OH). An internal volume of H. halobium envelope vesicles of 2.9 µL/mg of protein28 was used in this study. RESULTS AND DISCUSSION Response Characteristics of the Electrodes. To obtain the most suitable electrode, we examined the effects of solvent mediators on response to triethylammonium, because solvent mediators have been shown to markedly affect the response characteristics of the electrodes based on NaHFPB.10,11 Calibration graphs were obtained by measuring known amounts of triethylamine hydrochloride added to a solution containing 150 mM NaCl and 10 mM NaH2PO4/Na2HPO4 (pH 7.5) and plotting the concentrations against the obtained corresponding emf values. The solvent mediators tested were benzyl 2-nitrophenyl ether, 2-fluoro2′-nitrodiphenyl ether, o-nitrophenyl octyl ether, dioctyl phthalate, bis(2-ethylhexyl) sebacate, tris(2-ethylhexyl) phosphate, and tricresyl phosphate. Among them, both benzyl 2-nitrophenyl ether and 2-fluoro-2′-nitrodiphenyl ether afforded higher degrees of sensitivity to triethylammonium; however, the potential stability of benzyl 2-nitrophenyl ether was significantly superior to that of 2-fluoro-2′-nitrodiphenyl ether. Thus, we used benzyl 2-nitrophenyl ether in the present experiments. The electrode using benzyl 2-nitrophenyl ether exhibited a near-Nernstian response to triethylammonium in the concentration range of 5 × 10-6-1 × 10-2 M with a slope of 58.5 mV per concentration decade (Figure 1a). The lower limit of detection was 1 µM. We further made a calibration graph in a solution containing 4 M NaCl and 10 mM NaH2PO4/Na2HPO4 (pH 7.5) (Figure 1b). It should be emphasized that the electrode still showed a high degree of sensitivity toward triethylammonium even in the presence of an extremely high concentration of Na+ and gave a slope of 58.0 mV per concentration decade with a detection limit of 6 µM. Thus, the electrode (25) Kamo, N.; Racanelli, T.; Packer, L. Membr. Biochem. 1982, 4, 175-188. (26) Hazemoto, N.; Kamo, N.; Kondo, M.; Kobatake, Y. Biochim. Biophys. Acta 1982, 682, 67-74. (27) Lanyi, J. K.; MacDonald, R. E. Methods Enzymol. 1979, 56, 398-407. (28) Kamo, N.; Takeuchi, M.; Hazemoto, N.; Kobatake, Y. Arch. Biochem. Biophys. 1983, 221, 514-525.
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Figure 1. Calibration graphs for the electrode with triethylammonium in buffer solution containing (a) 150 mM NaCl and 10 mM NaH2PO4/ Na2HPO4 (pH 7.5) and (b) 4 M NaCl and 10 mM NaH2PO4/Na2HPO4 (pH 7.5). Pota Table 1. Selectivity Coefficients, log ki,j
interfering ion (j ) Mg2+ Ca2+ Li+ Na+ K+ a
log
kPot i,j
-5.4 -5.2 -4.1 -3.8 -3.4
interfering ion (j ) +
NH4 CH3NH3+ choline (CH3)4N+ (C2H5)4N+
log
kPot i,j
-3.5 -2.8 -1.0 -0.2 1.3
i is triethylammonium and j is the interfering ion.
can also be used even in the presence of such high Na+ concentrations. The response time (90% final signal) of the electrode was less than 10 s when the concentration of triethylammonium was changed from 50 to 100 µM in both solutions containing 150 mM NaCl and 4 M NaCl buffered with 10 mM NaH2PO4/Na2HPO4 (pH 7.5). The selectivity coefficients of the electrode are given in Table 1. The electrode showed no significant interference from inorganic cations such as Na+ and K+. However, the response to lipophilic quaternary ammonium ions such as (C2H5)4N+ was greater than that to triethylammonium, because the selectivity of the electrode using the ion-exchanger was determined by the order of the lipophilicity of the organic ammonium ions.10,11 The pH dependence of the electrode at three triethylammonium concentrations is shown in Figure 2. The pH dependence was measured in 150 mM NaCl, and the pH of the solution was changed by adding an appropriate amount of dilute hydrochloric acid or sodium hydroxide solution. The response of the electrode was independent of pH in the range of 6-10. The decrease in the potential above pH 10 was attributable to an increase in the concentration of the unprotonated form of triethylamine, because the pKa value of triethylammonium was reported to be 10.7.14 Thus, the electrode 1852 Analytical Chemistry, Vol. 73, No. 8, April 15, 2001
Figure 2. Effects of pH on electrode emf response at three triethylammonium concentrations in the presence of 150 mM NaCl. The pH of the solution was changed by adding an appropriate amount of dilute hydrochloric acid or sodium hydroxide solution.
was applicable over a wide pH range including the present range of 7.5-9.6. ∆pH Measurements for Liposomal Membranes. First, we examined whether the triethylammonium electrode could be applied to determine the pH difference across liposomal membranes composed of egg PC and cholesterol, which was successfully determined by the methylammonium electrode reported previously.5 As the spontaneous diffusion rates of H+ and/or OHacross liposomal membranes are rather low,29-31 it was assumed that the inner pH of liposomes would be constant during the short period of the experiments. Thus, it was expected that when the outer pH was made more alkaline than the inner liposomal pH, the uptake of triethylammonium in liposomes would be induced. As shown in Figure 3, when sodium hydroxide was added to the liposome suspension to make pHin < pHout, a significant decrease in electric potential and a corresponding decrease in triethylammonium concentration in the outer medium were observed, and further addition of sodium hydroxide caused further accumulation of triethylammonium inside the liposomes. The accumulated triethylammonium in the liposomes was released when the outside pH was returned to the initial value. This result clearly showed that the triethylammonium electrode can monitor changes in triethylammonium concentration caused by transmembrane pH differences. It is reasonable to consider that the accumulation of triethylamine inside liposomes did not affected inner pH, because the buffer capacity of the inner medium of the liposomes was high and the initial triethylamine concentration was low. We calculated the ∆pH from eq 3 and examined its dependence on the external pH. The intravesicular volume of liposomes used in this study was estimated as 11.8 µL/mg of lipid by a spin label (29) Deamer, D. W.; Gutknecht, J. Methods Enzymol. 1986, 127, 471-480. (30) Blume, A. Methods Enzymol. 1986, 127, 480-486. (31) Cafiso, D. S. Methods Enzymol. 1986, 127, 502-510.
Figure 3. Monitoring of changes in emf with variations in the external pH. Liposomes composed of egg PC/cholesterol were suspended in a solution (500 µL) containing 100 µM triethylamine hydrochloride, 150 mM NaCl, and 10 mM NaH2PO4/Na2HPO4 (pH 7.5) at 3.8 mg of lipid/mL. At the times indicated by the first and second arrows, 3 and 2 µL, respectively, of 160 mM NaOH were added. The third arrow indicates the time at which 5 µL of 160 mM HCl was added to reestablish the initial pH. The pH values of the solution after addition of NaOH and HCl were monitored simultaneously using a miniaturized pH glass electrode and are shown on the curves.
Figure 4. Dependence of ∆pH across the liposomal membrane on external pH. The sign of ∆pH on the ordinate was changed to negative to illustrate the differences (pHout - pHin). The external pH shown on the abscissa was measured using a glass pH electrode.
method. This corresponded to an inner volume (v) of liposomes and an outer medium volume (V) of 22.5 and 477.5 µL, respectively. Using these values and electric potential changes, we calculated ∆pH and found a strong linear correlation between ∆pH and external pH, as shown in Figure 4. We plotted -∆pH as the ordinate, to highlight differences (pHout - pHin) and represent increasing external pH as a positive value. A slope of 1 means that any change in pHout yields an equal change in -∆pH. Linear regression analysis revealed that the slope and the intercept of the line were 0.968 and 0.032, respectively (r ) 0.996; n ) 11). We further analyzed the correlation between -∆pH and external pH using two liposomes prepared from egg PE/egg PG and lipid extracted from E. coli. Linear regression analysis using liposomes composed of egg PE/egg PG measured in the external pH range (7.5-9.1) showed a slope of 0.968 and intercept of 0.050 (r ) 0.996; n ) 11), while that using liposomes composed of E. coli lipid measured in the external pH range (7.5-9.3) showed a slope of 0.989 and intercept of 0.060 (r ) 0.996; n ) 11). These results indicated that the triethylammonium electrode was quite suitable
Figure 5. Monitoring of changes in emf with variations in the external pH. E. coli cells were suspended in a solution (500 µL) containing 100 µM triethylamine hydrochloride, 150 mM NaCl, 10 mM sodium lactate, and 10 mM NaH2PO4/Na2HPO4 (pH 7.6) at 12.8 mg of cell protein/mL. At the times indicated by the first to fourth arrows, 1 µL of 1 M NaOH was added. The fifth arrow indicates the time at which 4 µL of 1 M HCl was added to reestablish the initial pH. The pH values of the solution after addition of NaOH and HCl were monitored simultaneously using a miniaturized pH glass electrode and are shown on the curves.
for determining ∆pH across liposomal membranes irrespective of the kind of liposomes and will be effective as an alternative to tracer-labeled methylamine, which has been used extensively to date,6-9 to determine the internal pH of cells (pHin) through the measurement of ∆pH and the external pH (pHout) of the medium using a pH electrode. ∆pH Measurements for E. coli Cells. Then, we applied the method to examine the external pH dependence of ∆pH of E. coli cells. Previously, we used a methylammonium electrode for this measurement.5 However, the uptake of methylammonium into E. coli cells could not be measured directly, because a large amount of K+ efflux was induced from E. coli cells when the pH of the medium was made alkaline,32 and this K+ efflux seriously interfered with the electrode response.5 The selectivity coefficient of the methylammonium electrode toward K+ (log kPot i,j ) -1.1) was not sufficient to allow measurement of methylammonium uptake in the presence of a large amount of K+.5 In contrast, the present triethylammonium electrode showed remarkably high selectivity against K+ (log kPot i,j ) -3.4), and this seemed to be sufficient to measure the external pH dependence of ∆pH of E. coli cells even in the presence of a large amount of K+. As shown in Figure 5, when sodium hydroxide was added to the E. coli cell suspension to make pHin < pHout, a decrease in electric potential and a corresponding decrease in triethylammonium concentration in the outer medium were clearly observed. However, further addition of large amounts of sodium hydroxide caused the gradual efflux of triethylammonium accumulated in the E. coli cells, leading to an increase in triethylammonium concentration in the outer medium. When the outside pH was returned to the initial pH by adding hydrochloric acid, the electric potential returned to the initial value. To discuss these external pH-dependent ∆pH changes in E. coli cells with regard to cellular membrane function, changes in the internal pH of the cells (pHin) with variations in the external (32) Yamasaki, K.; Moriyama, Y.; Futai, M.; Tsuchiya, T. FEBS Lett. 1980, 120, 125-127.
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Figure 6. Changes in the internal pH of E. coli cells (pHin) with variations in the external pH (pHout).
pH of medium (pHout) were plotted (Figure 6). The pHin values were calculated using the relation of pHin ) ∆pH + pHout. As can be clearly seen in Figure 6, the intracellular pH of the cells was rather constant at around 7.6-7.8 over the range of extracellular pH from 7.6 to 8.5. When the extracellular pH was above 9, however, the intracellular pH increased rapidly and exceeded 8. This profile of the pH dependence of E. coli cells was in accordance with that observed previously with tracer-labeled methylamine.21,22 The increase in pHin under higher pHout suggested that a significant amount of H+ permeates through E. coli membranes, in contrast to artificial liposomal membranes. As discussed previously,5 in biological membranes there are many ion pathways via membrane proteins. Ion movement through the membrane proteins is important for the function of cell membranes. This membrane “leakage” must be successfully controlled to keep the intracellular pH neutral even in alkaline environments. The present results showed that E. coli intracellular pH regulation is successful up to around pH 8.5, and then it gradually diminishes at more alkaline pH, probably due to retarded membrane functions. Light-Induced ∆pH Changes in H. halobium Envelope Vesicles. Since the present electrode was highly sensitive even in 4 M NaCl, we applied the method to measure light-induced ∆pH changes in the envelope vesicles of H. halobium. The envelope vesicles of H. halobium have two representative lightreactive pigments: halorhodopsin, which is an inward-directed light-driven Cl- pump, creating in turn passive H+ uptake, and bacteriorhodopsin, which expels protons from inside to outside upon illumination.33-35 First, we used the strain KY-4, which has halorhodopsin alone.26,28 Halorhodopsin elicits subsequent H+ uptake into the cells upon illumination, resulting in pHin < pHout.28 Thus, it was expected that the uptake of triethylammonium inside cells would occur upon illumination. Indeed, potential changes corresponding to the uptake of triethylammonium were clearly observed as shown in Figure 7a. We calculated the light-induced ∆pH from eq 3. Using the intravesicular volume of KY-4 estimated as 2.9 µL/mg of protein28 and electrical potential change, we (33) Schobert, B.; Lanyi, J. K. J. Biol. Chem. 1982, 257, 10306-10313. (34) Lanyi, J. K. Physiol. Rev. 1990, 70, 319-330. (35) Oesterhelt, D.; Tittor, J. Trends Biochem. Sci. 1989, 14, 57-61.
1854 Analytical Chemistry, Vol. 73, No. 8, April 15, 2001
Figure 7. Light-induced ∆pH changes in the envelope vesicles of the strains KY-4 (a) and S9 (b) of H. halobium in a solution (500 µL) containing 100 µM triethylamine hydrochloride, 4 M NaCl, and 10 mM NaH2PO4/Na2HPO4 (pH 7.5) at concentrations of 13.6 and 14.6 mg of protein/mL, respectively. The light intensity was 750 W/m2.
calculated ∆pH as 0.8 under the present conditions, which agreed well with that reported previously.28 Then, we used strain S9, which has both halorhodopsin and bacteriorhodopsin.26,28 It has been experimentally established that when S9 vesicles are illuminated in 4 M NaCl at around neutral pH, rapid acidification (proton extrusion due to the action of bacteriorhodopsin) occurs in the outer medium, which is followed by prolonged alkalinization (proton uptake elicited by the action of halorhodopsin) in the medium.28 We confirmed this complicated pH change using the triethylammonium electrode. As shown in Figure 7b, the initial rapid acidification (due to the action of bacteriorhodopsin) in the outer medium caused a small increase in the concentration of triethylammonium released from vesicles initially containing 100 µM triethylammonium. Then, a gradual decrease in the concentration of triethylammonium, which corresponded to the uptake of triethylammonium inside cells induced by alkalinization in the outer medium (due to the action of halorhodopsin and the succeeding passive H+ uptake), was clearly observed. This profile was in good accordance with the results obtained previously with a pH glass electrode3 and the spin probe 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl.28 CONCLUSIONS The present approach can be applied to ∆pH measurements in various cell membranes, because the triethylammonium electrode showed no significant interference from Na+ and K+ present in large amounts in biological systems. This method is a new fundamental technique for estimating ∆pH in artificial and biological membranes. ACKNOWLEDGMENT This work was supported by the Okayama Foundation for Science and Technology, the Wesco Science Promotion Foundation and the Japan Society for the Promotion of Science. Received for review September 12, 2000. Accepted January 31, 2001. AC001090T
